PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is a devastating brain tumor lacking effective treatments. This is largely due to invasion of GBM cells, which enables escape from resection and drives inevitable recurrence, typically 2 cm from the location at diagnosis. Progress in developing therapies to combat this process has been slow due to problems with the cells being studied and the methods of analysis. First, existing studies have failed to recognize that infiltrating GBM cells extending beyond the tumor edge have evolved a unique adaptive cellular machinery due to local stressors in their microenvironment. Unfortunately, these cells at the invasive tumor front are often not the ones sampled in studies analyzing banked tumor tissue, which is typically procured from the readily accessible central portions of the tumor. Another problem is that most studies of invasiveness have used two- dimensional (2D) culture systems coated with a thin layer of ECM proteins, which fail to capture the dimensionality, mechanics, and heterogeneity of GBM invasion. To address these limitations, our team has developed intriguing data using site-directed biopsies from GBM and has tissue engineered platforms to study invasion in vitro. Using site-directed biopsies, we have shown increased GBM cell invasiveness and increased expression of invasion-promoting integrins and extracellular matrix (ECM) splice variants outside versus inside enhancing MRI regions. We have also developed patient-derived xenografts (PDXs) from these site-directed biopsies that exhibit more invasiveness when arising from the tumor edge. Our team also became among the first to bioengineer 3D hydrogel systems as a discovery platform In GBM. We found that, as CD44-mediated peritumoral invasion falls, perivascular integrin-based motility increases. Here, we will build upon this intriguing data by investigating our central hypothesis that as GBM cells exit the tumor core, reciprocal interactions with the microenvironment drive a targetable transition from peritumoral to perivascular invasion. These goals will be accomplished through three aims: Aim 1 ? Define changes in the tumor microenvironment promoting invasive change as tumor cells egress away from the central core to the outer edge of GBM; Aim 2 - Refine bioengineered culture models to replicate the microenvironment of the outer edge of GBM and identify the role of TAMs in driving invasion in this region; and Aim 3 - Define the role of integrins in the invasiveness of GBM cells from the outer tumor edge and identify druggable mediators of invasion in this region. To accomplish these goals, we will use novel PDXs and tissue engineered platforms, along with CRISPRi, single-cell technology, and site-directed biopsies. Our studies will discover novel mechanisms by which tumor cells and their microenvironment are altered to drive increased invasiveness as cells migrate away from the tumor core. This work will challenge conventional thinking by showing how GBM integrates distinct regional microenvironments. We will account for these distinctions when identifying novel druggable targets to disrupt GBM cell invasiveness, with potential applicability to other invasive cancers as well.